Mutationally activated BRAF is expressed in a wide variety of human cancers with a strikingly high prevalence in melanoma. Mutated BRAF promotes sustained activation of the RAF->MEK->ERK MAP kinase signaling pathway that in turn contributes to many of the aberrant behaviors of the melanoma cell. Using conditionally active forms of BRAF, it has been demonstrated that BRAF->MEK->ERK signaling can suppress apoptosis in a variety of mammalian cells. These data are consistent with the embryonic lethal phenotype of 5/WFnullizygous mice that display a variety of apoptotic defects. BCL-2 family proteins, comprising both pro- and anti-apoptotic members, play an essential role in the regulation of apoptosis. Hence, we find it highly provocative that we have identified a direct biochemical connection between activation of BRAF and the phosphorylation of the BH3 domain only protein BIM, a pro-apoptotic member of the BCL-2 family that plays an essential role in melanocyte apoptosis. Here we propose experiments to test the hypothesis that BRAF activation can influence apoptosis in normal melanocytes and in melanoma cells through alterations in BIM phosphorylation. To test this hypothesis we will utilize a system for the conditional activation of BRAF signaling in melanocytes. This system will be used to explore the global effects of BRAF activation in melanocytes as well as the specific effects of BRAF on apoptosis. To complement these studies, we will use genetic (RNAi) and pharmacological inhibitors of BRAF->MEK->ERK signaling or BIM expression to assess the importance of this pathway in melanocyte/melanoma cell apoptosis in response to trophic factor deprivation, matrix detachment or DNA damage. Although there is strong genetic and biochemical evidence for an important role for BRAF signaling in the initiation and progression of human melanoma, and for BIM in the regulation of melanocyte survival, we will test the hypothesis that these proteins are linked in a direct biochemical pathway. We shall elucidate the biochemical mechanism(s) by which BRAF regulates the proapoptotic effects of BIM in mammalian cells with an emphasis on the role of phosphorylation in regulating BIM expression/activity. We will characterize the extent of BRAF-induced BIM phosphorylation, identify the specific sites and analyse the heterogeneity of BIM phosphorylation in response to BRAF activation. We shall confirm these sites by expression of normal and mutated BIM and by the generation of phospho-specific antisera. Finally, we will use biochemical and genetic techniques to explore the mechanism by which BRAF-induced phosphorylation inhibits BIM's pro-apoptotic activity. We believe that the proposed experiments will provide a general system for the analysis of the earliest effects of BRAF in the conversion of normal melanocytes into metastatic melanoma cells. Moreover, these studies will lead to a better understanding of the role(s) of BRAF and BIM in controlling apoptosis in mammalian cells. Finally, we anticipate that these studies will ultimately lead to the design and implementation of new therapeutic strategies to target metastatic melanoma cells for apoptosis leading to remission of this dread disease.